This subproject will utilize genetic studies in mice to help identify new genes and pathways that are involved in the development of atherosclerotic lesions and inflammation. During the past grant period, this subproject 6 has focused on the analysis of naturally occurring variations in three separate genetic crosses, involving strains C3H/HeJ (C3H), CAST/Ei (CAST), and DBA/2J (DBA), each crossed to the atherosclerosis-susceptible C57BL/6J (B6) strain. These studies have revealed novel genetic factors for atherosclerosis involving vascular cells, monocyte/macrophages, and lipoprotein metabolism. We now propose to extend these studies in the same 3 sets of strains to address the following 3 mechanistic questions relating to the genetic determinants of atherosclerotic lesion development and lesion composition, with a focus on lipid oxidation, inflammation, and calcification. (1) How do oxidized lipids induce expression of inflammatory genes, and how do genetic variations in these pathways influence the development of atherosclerotic lesions? For these studies, we will focus on the C3H x B6 cross. In this model, we have produced strong evidence that the genes that control response of endothelial cells to oxidized lipids are the primary determinant of atherosclerosis susceptibility in this model. We will now identify the pathways and genes involved using in vitro and in vivo approaches. (2) What genetic factors contribute to the ability of HDL to protect against oxidative stress and inflammation? For this, we will focus on the CAST x B6 cross, for with multiple genetic loci controlling HDL levels and/or function have been isolated as congenic strains. The genes responsible will be identified and the functional properties defined in conjunction with the subproject by Fogelman. (3) What are the genetic factors contributing to vascular calcification, and what are the interactions between atherosclerosis, vascular calcification, myocardial calcification and bone metabolism? These aspects will be studied using the DBA x B6 cross. Loci on chromosomes 2 and 6 have been identified as influencing both bone density and lipid metabolism, a locus on chromosome 7 (Dyscalc) influences cardiovascular calcification and a locus on chromosome 10 influences atherosclerosis. Positional candidate genes in these regions will be investigated, and interactions between the loci will be studied.